Gene activation and silencing are controlled at the level of chromatin. Regions of transcriptionally silent heterochromatin are dynamic, and without active intervention, will spread into areas of euchromatin resulting in gene silencing. Barrier insulator elements function to maintain the boundaries between euchromatin and heterochromatin and are essential for maintaining higher order chromatin structure to regulate appropriate patterns of gene expression. To understand how our genome is regulated, a thorough understanding of barriers insulators is necessary. Perturbations in this critical regulatory mechanism contribute to the abnormalities in gene expression observed following chromosomal translocations in both inherited genetic disease and cancerous states. In addition, mutations disrupting barrier insulator function have been linked to inherited hemolytic anemia. The mechanisms underlying these aberrations in gene expression are unclear, because little is known about the structure and function of mammalian barrier insulators. The goal of this proposal is to characterize the location, structure, and function of barrier insulator elements in human cells. The goal of specific aim 1 of this proposal is to identify a molecular signature associated with barrier insulator elements. We hypothesize that this signature will be composed of the USF proteins, their associated histone methyltransferases (PRMT1, PRMT4, SET7/9), and acetyltransferases (CBP, P300, PCF), as well as an active chromatin structure (H3Ac, H4Ac, H3K4me2). We will utilize chromatin immunoprecipitation coupled with ultrahigh throughput Solexa sequencing (ChIP-seq) to create genome-wide maps of barrier insulator- associated factor binding and chromatin architecture in primary nucleated human erythoid cells. Regions of barrier protein co-occupancy will be identified and selected sites subjected to position effect variegation (PEV) assays to confirm that these regions represent functional barrier insulators. The goal of the second specific aim of this proposal is to gain a comprehensive understanding of barrier insulator function in mammalian cells through detailed functional characterization of a previously identified group of potential barrier insulators, located in a focus group of erythrocyte membrane protein genes. We hypothesize that barrier insulator activity is cell-type specific and that the USF proteins are necessary for insulator function. To gain insights into the cell- type specificity of insulator structure and function, candidate barrier insulators will be subjected to quantitative ChIP analyses and PEV assays in both erythroid and non-erythroid cells, and selected sites will be studied in detail in transgenic mouse models. We will also employ shRNA knock-down of the USF proteins to determine if they are necessary for barrier insulator function in mammalian cells. The combination of genome-wide technologies and detailed functional analyses applied to the study of barrier insulators will provide valuable mechanistic insights into this fundamental mechanism of gene regulation.